Comparison of different regimens of estradiol benzoate treatments followed by long-acting progesterone to prepare noncycling mares as embryo recipients.

The present study evaluated the influence of different regimens of estradiol benzoate (EB) treatments followed by a single dose of long-acting progesterone (LA P4) on plasma estrogen and P4 concentrations in noncyclic mares prepared as embryo recipients. Twenty-one anestrous mares were distributed into three groups (n = 7 mares per group), according to the EB dose received (single dose of 2.5 mg, total of 5 mg in decreasing doses, and total of 10 mg in decreasing doses), which was followed by a single administration of 1500 mg of LA P4 in all groups. Mares were reevaluated during the ovulatory phase and seven of them became part of the cyclic nontreated control group. Ultrasonography was performed to monitor endometrial edema, and blood samples were collected to measure estradiol (E2), estrogen conjugate (EC), and P4 by RIA. Maximum uterine edema was achieved 24 hours after administration of EB in all treated groups. Maximum E2 concentrations were observed 24 hours after the first EB injection in treated groups and there were no differences (P > 0.05) among treatments. Maximum EC concentration was observed 24 hours after the single EB injection in the 2.5-mg group, whereas in the 5- and 10-mg groups EC peaks were observed 48 hours after the first EB administration. Maximum P4 concentrations were detected 24 hours after LA P4 injection, although higher P4 concentrations were observed in the group treated with 2.5 mg of EB than in that treated with 10 mg of EB (P < 0.05). Because P4 concentrations were reduced after administration of high doses of EB, we also measured 17α-hydroxyprogesterone (17-OH-P) to test the hypothesis that high concentrations of EB would accelerate the conversion of P4 to 17-OH-P. However, 17-OH-P concentrations paralleled P4 profile in all groups, irrespective of EB doses. In summary, the three EB treatment regimens induced similar E2 peaks, although the observation of EC peaks 24 hours after E2 peaks in the 5- and 10-mg groups indicate that an excess of E2 was given, which was converted into EC to be inactivated. Administration of 10 mg of EB reduced P4 concentrations 24 hours after LA P4 was given. We demonstrated that the mechanism by which this reduction occurred was not by an increase in P4 metabolism to 17α-OH-P. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 appears to be a more appropriate regimen to treat noncyclic mares, although additional studies are needed to verify embryo survival with this treatment dose.

Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares.

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches.

Stimulation of Sertoli cell proliferation: defining the response interval to an inhibitor of estrogen synthesis in the boar.

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11-16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.

Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: induction of ovulation, hormone profiles, and inter-ovulatory intervals.

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17beta (E(2)), and progesterone (P(4)) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.

Reducing estrogen synthesis does not affect gonadotropin secretion in the developing boar.

Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species.

Endocrine and paracrine control of sperm production in stallions.

The specific nature and relative contribution of the major hormones involved in regulation of reproductive function of the stallion are not well defined nor have paracrine or autocrine factors been identified. Over the last 12 years, our laboratory has been engaged in characterizing the hypothalamic-pituitary-testicular axis (HPT) in stallions. A number of endocrine factors and mechanisms important for normal reproductive function have been investigated. Studies investigating poor fertility in stallions suggest that a closer look at the testicular level is warranted. For a complete understanding of intratesticular control mechanisms including cell-to-cell interactions in the stallion, studies on the actions of paracrine/autocrine factors such as growth factors, inhibin, activin, and oxytocin are needed. In other species, paracrine/autocrine systems appear to be important in modulating endocrine control of testicular function and spermatogenesis.

Maternal-conceptus signalling during early pregnancy in mares: oestrogen and insulin-like growth factor I.

Embryonic production of oestrogen is thought to play an important role in conceptus-maternal signalling during early pregnancy in mares, and may be regulated in an autocrine or paracrine fashion by insulin-like growth factor I (IGF-I). In this study, the hypothesis that IGF-I stimulates embryonic oestrogen synthesis, which in turn stimulates uterine IGF-I secretion was tested. Specific sources of IGF-I in the uterine lumen were characterized. Preimplantation embryos, uterine biopsies, and uterine flush fluids were collected on day 13 of pregnancy. Embryos were cultured whole for 24 h, or dispersed and incubated in serum-free culture medium supplemented with androstenedione or testosterone (0-10 microg ml(-1)) and IGF-I (0-100 microg ml(-1)). Oestrogen synthesis was increased by addition of androgen, but there was no dose-dependent effect of IGF-I. Endometrial explants were cultured for 24, 48 and 72 h in serum-free medium supplemented with oestradiol. IGF-I was measured by radioimmunoassay in embryo-conditioned medium, explant culture medium, blastocoelic fluid, concentrated (x 100) uterine flush fluid and endometrial-tissue homogenate. Both the embryo and endometrium produced significant quantities of IGF-I, indicating a role for this growth factor in autocrine-paracrine signalling during early pregnancy. However, secretion of IGF-I by endometrial explants was not modulated by oestrogen.

Testicular biopsy in stallions: diagnostic potential and effects on prospective fertility.

Testicular biopsy was performed as a standing procedure on fertile, mature stallions (n=7) under sedation and local anaesthesia. The 'Biopty' instrument was used to collect three samples from the left testis of each stallion. The oestradiol, testosterone and inhibin content in each testis were assessed by validated radioimmunoassay (RIA); protein concentrations were determined and the testes were prepared histologically for haematoxylin and eosin staining. The fertility status of each stallion was assessed before and after testicular biopsy by semen evaluation, changes in total scrotal width, pregnancy rate per oestrous cycle, serum and seminal plasma antisperm antibody concentrations, and determination of plasma LH, FSH, oestradiol, testosterone and inhibin concentrations once a week by validated RIA. Testicular testosterone (3.85 +/- 1.24 ng mg(-1) protein), oestradiol (3.01 +/- 0.95 ng mg(-1) protein) and inhibin (11.06 +/- 1.69 ng mg(-1) protein) content were measured successfully from tissue samples by RIA. Active spermatogenesis was evident in all histological samples. The mean numbers of progressively motile morphologically normal spermatozoa were not significantly different in the last three ejaculates of the 7 day periods of semen collection before (3.37 +/- 0.48 x 10(9) spermatozoa per ejaculate) and after (3.77 +/- 0.48 x 10(9) spermatozoa per ejaculate) testicular biopsy. Total scrotal width varied within normal ranges throughout the year (from 11.7 +/- 0.3 to 10.0 +/- 0.4 cm). Pregnancy rates per oestrous cycle before (50 +/- 6%) and after (63 +/- 9%) testicular biopsy were not significantly different. The concentration of antisperm antibodies in serum and seminal plasma samples did not vary significantly before and after testicular biopsy. Plasma LH, FSH, oestradiol, testosterone and inhibin concentrations fluctuated within normal ranges after biopsy in the expected seasonal pattern. In conclusion, the present study demonstrates that using the 'Biopty' instrument to obtain testicular biopsy samples from stallions has no apparent detrimental effects on prospective stallion fertility and can be used to evaluate testicular factors and structure.

Pituitary response to steroid replacement therapy in fertile, subfertile and infertile stallions after castration.

Recent studies in our laboratory investigating plasma gonadotrophin concentrations before and after castration indicate that the hypothalamic-pituitary axis is not the original site of dysfunction in stallions with idiopathic infertility. In the present study, fertile, subfertile and infertile stallions were subjected to replacement therapy with oestradiol and testosterone 1 year after castration to investigate hypothalamic-pituitary axis function further. Fertile (n=5), subfertile (n=3) and infertile (n=2) castrated stallions of Light horse breeds, aged 11-23 years, were given either oestradiol cypionate or testosterone propionate for 28 days during the breeding season. Oestradiol cypionate in cottonseed oil (30 mg; 6 mg ml(-1)) was administered i.m. at 2 day intervals starting in May for 28 days. Eight weeks after the last oestradiol cypionate injection, testosterone propionate in vegetable oil (85 mg; 8.5 mg ml(-1)) was administered i.m. at 2 day intervals for 28 days. Heparinized blood samples were taken from the jugular vein three times a week from 3 weeks before the start of oestradiol cypionate treatment until 4 weeks after the last testosterone propionate injection. Saline or 25 microg GnRH in 1 ml saline were administered to the stallions on days 24 and 25, respectively, of the 28 day steroid treatments. Jugular blood samples were collected periodically through a catheter from 60 min before saline or GnRH injection until 420 min after injection. The plasma samples were stored at -20 degrees C until analysed for plasma LH, FSH, oestradiol and testosterone concentrations by validated radioimmunoassay. Exogenous steroid treatment increased plasma oestradiol and testosterone concentrations to concentrations close to normal circulating values (oestradiol: 49 pg ml(-1); testosterone: 1.3 ng ml(-1)) in all the groups. There were no significant differences in plasma LH or FSH concentrations among the groups during any of the treatment periods. Oestradiol treatment significantly increased (P < 0.05) LH concentrations, whereas testosterone treatment decreased LH concentrations, although the difference was not significant. Oestradiol treatment significantly decreased FSH concentrations (P < 0.05), whereas testosterone treatment significantly increased FSH concentrations (P < 0.05). There were no differences in LH and FSH responses to GnRH challenge among the three groups of stallions. In conclusion, the results of the present study indicate that the hypothalamic-pituitary axes in these subfertile and infertile stallions were not the original sites of dysfunction. Further studies at the testicular level are required.

Uncoupling of the equine reproductive axes during transition into anoestrus.

The reproductive activity of light horse mares (n=12) was monitored each day from 3 September until 29 January, or until the mares entered anoestrus, by behaviour evaluation, ultrasonography and blood sampling. Follicles, corpora lutea and ovulation, as well as oestradiol, progesterone, and LH and FSH concentrations, were analysed to determine a reproductive profile for the transition into anoestrus. The results of the present study indicate that light horse mares progress through four phases during the autumn transition into anoestrus: (i) normal cycles; (ii) aberrant cycles; (iii) anovulation, with significant follicular activity; and (iv) anoestrus. One of the first changes observed was a progressive decrease in mean progesterone concentrations during normal cycles (summer: 7.1 +/- 0.4 ng ml(-1); last cycle: 3.6 +/- 0.2). Regression analysis indicates that large follicles progressively lose their ability to produce oestradiol in autumn (third last cycle: 8.5 +/- 1.3 pg ml(-1); last cycle: 5.7 +/- 0.5). During the last ovulatory surge, LH concentrations decreased approximately 50% in 6 of 8 mares. The duration of the follicular phase increased with each cycle approaching anovulation. However, the diameters of the follicles ovulated did not differ. The duration of the luteal phase during ovulatory cycles did not change. Four of the 12 mares developed spontaneously prolonged corpora lutea and were eliminated from the analysis. An anovulatory follicular growth phase occurred immediately before anoestrus in 7 of 8 mares. FSH preceded follicular growth during all cycles and persisted throughout the anovulatory period. During anoestrus, plasma oestradiol, progesterone and LH concentrations remained at basal concentrations. FSH concentrations remained high in 3 of 8 mares but did not cause follicular growth. It is concluded that ovarian and pituitary events become uncoupled during the transition into anoestrus.

Effects of age, season, and fertility status on plasma and intratesticular immunoreactive (IR) inhibin concentrations in stallions.

The nature of the relationship between inhibin and reproductive function in the stallion is yet to be elucidated. Blood and testes from 51 light horse stallions ranging in age from 2 mo to 25 years were collected during the breeding and nonbreeding seasons to study the effects of testicular maturation, aging, season, and fertility status on peripheral and intratesticular concentrations of Ir inhibin and other reproductive hormones. Of the 51 stallions, 12 age-matched stallions (6 fertile, 3 subfertile, and 3 infertile) were used in the fertility study. Blood samples were taken before castration and plasma stored at -20 degrees C for analysis of Ir inhibin, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estradiol (E2), and estrogen conjugates (EC) by radioimmunoassay (RIA). Testes were homogenized and testicular extracts prepared and frozen at -70 degrees C for analysis of Ir inhibin, T, E2, and EC by RIA. Plasma concentrations of Ir inhibin, LH, FSH, T, E2, and EC and intratesticular concentrations of Ir inhibin, T, E2, and EC increased with age (P < 0.01). The most dramatic effect appeared to be during testicular maturation. An aging effect was not observed in adult stallions. A seasonal effect was not detected for any of the plasma hormones, whereas for the intratesticular hormones the only change noted was an increase in T in the nonbreeding season (P < 0.05). Plasma Ir inhibin, E2, and EC were lower (P < 0.01) and gonadotropins higher (P < 0.05) in infertile stallions. Plasma T levels did not change. Intratesticular Ir inhibin concentrations tended to be lower (P < 0.1) in subfertile stallions and significantly lower (P < 0.01) in infertile stallions, whereas intratesticular steroid levels were not different among the three groups. In conclusion, plasma and intratesticular Ir inhibin concentrations seem to be affected by testicular maturation and fertility status.

HCG binding to the testicular LH receptor is similar in fertile, subfertile, and infertile stallions.

Recent evidence in our laboratory suggests that the cause of idiopathic subfertility/infertility in breeding stallions may originate in the testes at the luteinizing hormone (LH) receptor or postreceptor level. The objective of this research was to determine if LH receptor binding activity is altered in subfertile and infertile stallions. Six fertile, three subfertile, and three infertile stallions, ages 11-23 years, were classified according to normal semen parameters and pregnancy rates and then castrated in the breeding season. Blood was collected prior to castration, and plasma was stored until analyzed for LH, follicle stimulating hormone (FSH), estrogen conjugates (EC), estradiol (E2), testosterone (T), and inhibin (I) by radioimmunoassay (RIA). Testicular cell membranes were prepared and snap-frozen until analyzed for LH binding activity by radioreceptorassay (RRA) using increasing amounts of I125 human chorionic gonadotropin (hCG). Luteinizing hormone receptor numbers and affinity constants were determined by Scatchard analysis. Plasma LH, FSH, EC, E2, and T levels did not differ between fertile and subfertile stallions, but LH and FSH were significantly higher (P < 0.05) and EC, E2, T, and I levels were significantly lower (P < 0.05) in infertile stallions as compared to fertile and subfertile stallions. Receptor number (Rt) and affinity constants Ka were similar (P > 0.05) between fertile (Rt = 9.44 x 10(-11) M, Ka = 0.300 x 10(10) M-1), subfertile (Rt = 13.02 x 10(-11) M, Ka = 0.194 x 10(10) M-1), and infertile (Rt = 7.65 x 10(-11) M, Ka = 0.380 x 10(10) M-1) stallions. In conclusion, these data suggest that an endocrine dysfunction in the testes of stallions with poor fertility may not be due to a LH receptor disorder but may be due to a postreceptor malfunction.

Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids.

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of surgical removal of endometrial cups on concentrations of chorionic gonadotrophin and subsequent fertility in the mare.

Seven pregnant mares underwent general anaesthesia, laparotomy, hysterotomy and removal of a 50-day conceptus. Eversion of the uterine horn through the hysterotomy site allowed direct visualisation and electrosurgical removal of endometrial cup tissue from 5 randomly selected mares (Nos 1-5), while cup tissue in 2 mares (Nos 6 and 7) was left intact. Two pregnant mares served as unoperated controls (Nos 8 and 9). Efforts to re-establish pregnancy were initiated 20 days after surgery. Serum samples collected before surgery and during the post-operative period were analysed for concentration of horse chorionic gonadotrophin (CG) by radioimmunoassay. Mean (+/- sd) weight and area of removed tissue was 9.91 +/- 4.6 g and 22.4 +/- 5.9 cm2, respectively. Concentrations of CG ranging from 3440 to 21,220 ng/ml were highest at the time of cup removal (50 days) and declined thereafter. Peak concentrations of CG at the time of surgery were not linearly correlated (r = -0.59) with mass of excised cup tissue. Individual CG half-life values for Mares 1-5 ranged from 5.4 to 8.8 days (mean 7.1 +/- 1.2 days). Peak CG concentrations occurred 68 days after conception with a rate of disappearance (half-time) of 13.4 +/- 0.4 days in Mares 6 and 7 and at 74 and 78 days after conception with a half-time of 14.0 +/- 5.7 days in Mares 8 and 9. Mares 1-5 exhibited behavioural oestrus and ovulation 30-44 days after surgery. Pregnancy occurred in 3 mares within 41 days after surgery and was associated with concentrations of CG below 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-response effects of gonadotropin-releasing hormone on plasma concentrations of gonadotropins and testosterone in fertile and subfertile stallions.

Five fertile and five subfertile stallions were treated with a single intravenous injection of saline the first week followed by a single intravenous injection of varying doses of gonadotropin-releasing hormone (5, 10, 25, 100, 500 micrograms) given in a randomized fashion over the next 5 weeks during the nonbreeding season. Blood samples were collected periodically before and after treatment for analysis of luteinizing hormone, follicle stimulating hormone, and testosterone content by radioimmunoassay. Before treatment, semen samples were collected every other day for 3 weeks for analysis of volume, concentration, motility, pH, and morphology. Basal plasma levels of luteinizing hormone were higher (P < 0.05) in the subfertile group, follicle stimulating hormone levels tended to be higher (P < 0.10) in the subfertile group, and testosterone levels were similar in the two groups. A significant linear-log dose-response relationship was observed for plasma luteinizing hormone (P < 0.05) and follicle stimulating hormone (P < 0.05) to exogenous gonadotropin-releasing hormone in both the fertile and subfertile group. A linear-log dose-response relationship was also observed for plasma testosterone (P < 0.05) in the fertile group. The magnitude of the luteinizing hormone and follicle stimulating hormone response to gonadotropin-releasing hormone across doses was similar in both groups of stallions. A significant testosterone response to gonadotropin-releasing hormone in the subfertile group of stallions was not observed (P > 0.05). Mean testosterone concentrations after treatment in terms of net increase and percent of baseline were significantly lower (P < 0.05) in the subfertile group compared to the fertile group.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal effects on seminal quality, plasma hormone concentrations, and GnRH-induced LH response in fertile and subfertile stallions.

Seasonal effects on hormonal and seminal parameters in subfertile stallions have not been well documented and could provide information that is needed to understand the underlying endocrine mechanisms associated with testicular dysfunction. Such information may be useful in developing diagnostic tools to identify those stallions who are candidates for treatment. This investigation characterizes and compares the effects of season on endocrine function and seminal quality in fertile and subfertile stallions. Eight fertile and six subfertile stallions between the ages of 5 and 18 years were injected intravenously once every hour for 3 hours with either 1 mL saline on the first experimental day or 5 micrograms gonadotropin-releasing hormone in 1 mL saline on the second experimental day during the nonbreeding and breeding season. Heparinized blood samples were collected periodically through a jugular catheter before and after treatment for analysis of luteinizing hormone, follicle-stimulating hormone, testosterone, and estrogen conjugates by radioimmunoassay. Semen samples were collected twice, 1 hour apart, from all stallions in both seasons for analysis of volume, concentration, motility, pH, and morphology. A series of low intravenous doses (5 micrograms) of gonadotropin-releasing hormone induced a significant luteinizing hormone response (P less than 0.05) compared with saline treatment in both fertile and subfertile stallions. Fertile stallions had a twofold higher (P less than 0.05) net increase in plasma luteinizing hormone levels (peak levels minus baseline levels) in the breeding seasons than in the nonbreeding season. The magnitude of the luteinizing hormone response relative to baseline levels in fertile stallions, however, was one-and-one-half times greater (P less than 0.05) in the nonbreeding season than in the breeding season. In contrast, season did not have an effect on the net increase in plasma luteinizing hormone or the magnitude of the luteinizing hormone response relative to baseline levels in subfertile stallions. The net increase in plasma luteinizing hormone was similar between the two groups of stallions in both seasons. The magnitude of luteinizing hormone response relative to baseline levels, however, was lower (P less than 0.05) in subfertile stallions (141 +/- 14%) than in fertile stallions (235 +/- 46%) in the nonbreeding season; the two groups exhibited similar responses in the breeding season. Compared with fertile stallions, subfertile stallions had twofold to fourfold higher (P less than 0.05) plasma levels of gonadotropins and similar testosterone levels. The number of total progressively motile sperm was lower (P less than 0.05) in subfertile stallions in both seasons.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.

Gonadotrophin and steroid concentrations in jugular and testicular venous plasma in stallions before and after GnRH injection.

Six normal stallions of light horse breeds aged 5-17 years were used from fall to winter to investigate the difference between steroid hormone concentrations in testicular and jugular venous blood before and after exogenous GnRH. At 48 h before experimentation, an indwelling cannula was placed surgically in the testicular vein of the stallion. After the stallion recovered from anaesthesia, a catheter was placed percutaneously in the jugular vein. Each stallion was housed in a tie stall to allow simultaneous sampling of jugular or testicular blood. On the first and second sampling days, respectively, 1 ml of physiological saline solution and a 1 ml solution of GnRH (25 micrograms) were administered intravenously. Samples were taken from both sites at intervals from 60 min before treatment to 780 min after treatment. Plasma was analyzed for luteinising hormone (LH) and follicle-stimulating hormone (FSH), 17 beta-hydroxyandrogens (androgens), oestrone and oestrogen conjugates by radioimmunoassay. Pre-treatment (baseline) plasma concentrations of both LH and FSH between jugular and testicular samples were similar. The difference between basal levels of jugular and testicular androgens, oestrone and oestrogen conjugates were 144-fold, 60-fold and 13-fold respectively, although individual variation was observed. A low dose of exogenous GnRH produced a significant LH and FSH response in testicular and jugular plasma (P less than 0.05). There were no significant changes in steroid secretion caused by the increases in LH and FSH (P greater than 0.05), although individual variation in the androgen response was apparent (P less than 0.1). There was a positive correlation between basal testicular venous androgen levels and the magnitude of the FSH response to GnRH (P less than 0.05). Significant correlations between baseline oestrogens and the magnitude of the gonadotrophin response was not observed. Surgery depressed jugular oestrogen conjugate values (P less than 0.001) when compared to pre-surgical samples. Spermatogenesis also was depressed (P less than 0.01) by surgical manipulation, although total viable spermatozoa counts returned to normal limits within 3-5 months post operatively. We developed a model that allows the study of dynamic endocrine events associated with the hypophyseal-gonadal axis of the stallion. Our findings confirm the presence of a testicular-jugular hormone gradient in the unanaesthetized stallion. We have demonstrated that a relatively low dose of GnRH can induce a significant gonadotrophin response and a variable androgen response, but not a significant oestrogen response. Although baseline levels of androgens and not oestrone and oestrogen conjugates appeared to affect pituitary responsiveness, other steroidogenic components may be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged pulsatile administration of gonadotrophin-releasing hormone (GnRH) to fertile stallions.

Hormonal effects of prolonged administration of gonadotrophin-releasing hormone (GnRH) were investigated in 7 fertile stallions in winter and summer. The stallions were divided into 4 groups so that 1 animal received 0.625 micrograms of GnRH and each of 2 animals received 1.25, 2.5 or 5.0 micrograms of GnRH subcutaneously every 30 min for 5 days. Daily blood samples were collected from 5 days before to 5 days after treatment for measurement of plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and oestrogen conjugates (EC). Five-minute blood samples were collected over a 4 h period prior to, and on the last day of, GnRH treatment in each season for analysis of pulsatile changes in plasma LH concentrations. Mean concentrations of LH and T prior to treatment were higher (P less than 0.05) in summer than winter, whereas plasma FSH and EC concentrations did not differ between seasons. Increasing the dose of GnRH appeared to increase plasma concentrations of LH, FSH and T above baseline in winter (P less than 0.05), whereas a significant effect was not observed in summer. Plasma EC concentrations were not affected by treatment in either season. In GnRH-treated stallions, plasma LH and T concentrations were positively correlated such that the profiles of these hormone concentrations appeared to change in a similar direction over time in winter (P less than 0.01) but not in summer. Plasma concentrations of FSH and EC were not correlated in either season. A positive correlation between plasma LH and FSH concentrations was detected in both winter (P less than 0.01) and summer (P less than 0.02). In contrast, changes in plasma LH and T and plasma FSH and EC concentrations were not correlated over time in either season in control stallions. A positive correlation between plasma LH and FSH concentrations in control stallions was observed in summer (P less than 0.03) but not in winter. Prior to treatment, pulse patterns of LH were similar in both seasons. Following administration of GnRH, irrespective of dose, pulse frequency increased (P less than 0.05) and pulse duration decreased (P less than 0.05) resulting in a decrease in the total amount of LH released per pulse (P less than 0.05) in winter only. Amplitude and interval were not affected by GnRH treatment in either season. The results of this study suggest that the pituitary gland and testes of fertile stallions are more responsive to administration of prolonged pulsatile GnRH in winter than in summer.